The objective of this research is to increase our understanding of the regulation of cholesterol metabolism via the interaction of low density lipoproteins (LDL) with cells. The primary focus of this research is on the interaction of LDL with its specific receptor on the plasma membrane of normal fibroblasts and on the alterations in the membrane of familial hypercholesterolemia (FH) cells which render this specific receptor function inoperable. Our aims are threefold. The first part is designed to identify the membrane receptor protein which binds LDL. Binding of LDL to this receptor is the first step in the uptake of LDL by the cell and leads in turn to regulation of endogenous cholesterol metabolism. The second part of this project is designed to characterize this protein at the submolecular level and to compare tryptic peptides of the LDL binding proteins synthesized by normal and FH cells in order to determine whether the defective binding of LDL in FH cells results from a point mutation, a frameshift mutation, or an alteration in post-translational modification. The third aspect of this research will be to utilize the LDL receptor purified on a polyacrylamide gel as an antigen for the formation of anti-LDL receptor antibody. Availability of a specific antibody to the binding protein will provide an important tool both for elucidation of the involvement of the receptor in the sequence of events occurring between the binding of LDL and the regulation of HMGCoA reductase and for the possible diagnosis and early detection of FH by radioimmunoassay. The long-term goals of this research are to understand the mechanism by which binding of a serum protein at specific sites on the cell surface induces the rapid uptake of that serum protein and regulates the activity and synthesis of intracellular proteins. A further goal is to demonstrate the applicability of the newly developed techniques of double label autoradiography on polyacrylamide gels and on thin layer chromatography plates to the detection and characterization of a genetic alteration in a non-enzymatic membrane protein. BIBLIOGRAPHIC REFERENCES: Gruenstein, Eric and Andrew Pollard (1976) "Double-Label Autoradiography on Polyacrylamide Gels with 3H and 14C" Analyt. Biochem. 76, 452-457.